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Robert S Molday, Laurie Molday; Molecular Characterization of Missense Mutations in Nucleotide Binding Domains of ABCA4 Associated with Stargardt Disease. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5392.
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ABCA4 is an ATP-binding cassette (ABC) protein that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to cytoplasmic leaflet of photoreceptor disc membranes. Mutations in ABCA4 are known to cause Stargardt disease (STGD1), an autosomal recessive disease characterized by the accumulation of bisretinoid compounds and a loss in vision. The purpose of this study is to determine the effect of disease-causing missense mutations in the nucleotide binding domains (NBDs) on the expression and functional activity of ABCA4 and correlate these properties with molecular modeling and disease.
Wild-type (WT) ABCA4 and disease-causing variants in NBD1 including T959I, N965S, T983A, T1019A, T1019M, S1063P, E1087D, E1087K, R1108C and variants in NBD2 including R2030Q, R2077W, and R2107P were generated by site-directed mutagenesis. ABCA4 variants were expressed in HEK293T cells and their level of expression was measured on Western blots after solubilization in CHAPS buffer. The basal and N-Ret-PE stimulated ATPase activities of immunoaffinity purified ABCA4 variants were measured using a Promega luminescence assay kit. The localization of the ABCA4 variants expressed in COS-7 cells was determined by confocal microscopy. The structure of NBD1 and NBD2 of ABCA4 was modeled after the cryo-EM structure of ABCA1.
Disease-associated ABCA4 variants expressed at or below levels of WT ABCA4 after detergent solubilization. As previously reported, detergent-solubilized WT ABCA4 showed a basal ATPase activity that was activated 2.0-2.5 fold by N-Ret-PE. Most disease-causing mutants showed reduced basal and N-Ret-PE activated ATPase activity. WT ABCA4 localized to large intracellular vesicles containing the ER marker calnexin in transfected COS7 cells. The disease mutants showed variable cellular localization ranging from large vesicles characteristic of WT ABCA4 to a reticular pattern representative of the ER. The expression and functional activity was used to provide an indicator of the severity of STGD1 associated with these mutations. Molecular modeling defined the localization of the variants within the NBD structures.
STGD1-associated ABCA4 variants generally showed reduced expression and functional activity, properties that can be used to estimate the effect of the mutations on the pathogenicity of STGD1.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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